Effect of 1-(chloromethyl)silatrane on the alteration of the blood elements during extracorporeal circulation

The damaging effect of cardiopulmonary bypass on blood elements and the possibility of its correction with 1-(chloromethyl) silatrane have been investigated. Cardiopulmonary bypass is a powerful damaging factor producing a wide range of effects including the activation of lipid peroxidation, reduction of erythrocyte membrane resistance to ultra-sound, destruction of blood elements and appearance of hemoglobin in the plasma (hemolysis). A possible mechanism of cardiopulmonary bypass damaging effect on blood elements is suggested. The use of 1-(chloromethyl)silatrane drastically increases the resistance of blood element membranes to injury, which seems promising for the drug application during cardiopulmonary bypass.     

Possible mechanisms of anti-ulcer action of silatranes

A stimulating effect of methylethyl(silatrane-I-ilmethyl)-sulphonium iodides, 1-(ethoxy)silatranes and 1-(chloromethyl)silatrane on the processes of rat test acetate ulcer healing is studied. Silatranes efficiency is not inferior to oxyferriscorbone and exceeds that of methacil. It is shown that all preparations inhibit the processes of lipids peroxidation in the blood, in liposomes and stomach tissue.     

Investigations on the mechanism of the hypocholesterolemic action of 1-ethoxysilatrane

Summary Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 -hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation.A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbant assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.     

Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals

Peroxiredoxin I (Prx I) is a key cytoplasmic peroxidase that reduces intracellular hydroperoxides in concert with thioredoxin. To study the role of tissue Prx I in protection from oxidative stress, we generated Prx I-/- mice by gene trapping. We then evaluated the acute-phase tissue damage caused by ferric-nitrilotriacetate (Fe-NTA). Increases in serum aspartate aminotransferase and alanine aminotransferase levels were significantly greater in Prx I-/- than wild-type mice, 4 and 12 h after the injection of Fe-NTA. Using real-time EPR imaging, we examined the reduction of the stable paramagnetic nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in vivo, and found that the half-life of this spin probe in the liver and kidney was significantly prolonged in the Prx I-/- mice. These results demonstrate that Prx I-/- mice have less reducing activity and are more susceptible to the damage mediated by reactive oxygen species in vivo than wild-type mice.     

Rhizobium japonicum derivatives differing in nitrogen-fixing efficiency and carbohydrate utilization.

Four derivatives of Rhizobium japonicum 110 were isolated on the basis of morphologically different colonies formed on yeast extract-mannitol-HM salts medium. All are able to nodulate Lee soybeans. The bacteria-plant associations formed by each clone have measurable acetylene-reducing activity, but those formed by two of these clones (designated L1-110 and L2-110) are 5- to 10-fold less efficient than those formed by the others (designated I-110 and S-110). These derivatives were not detectable with ordinary culture techniques since, because of cell adherence, genetically mixed colonies result. When a detergent (Tween 40 at 0.01%, vol/vol) was added to the dilution medium, separate clones resulted. The metabolic basis for the gross differences in colony morphology on yeast extract-mannitol-HM salts medium was found to be that L1-110 and L2-110 utilized p-mannitol for growth, whereas I-110 and S-110 did not. These clones differ analogously in ability to utilize D-arabitol. Clones I-110 and L1-110 were chosen for studies of growth rates on various carbohydrates. Although clone I-110 and clone L1-110 did not differ in growth rates on a number of sugars, such as gluconate, arabinose, glycerol, and mannose, they differed in growth rates on glucose and fructose. Although clone I-110 grew faster on glucose than did clone L1-110, clone L1-110 grew faster on fructose than did clone I-110. Clones I-110 and L1-110 showed identical responses to several antibiotics and deoxyribonucleic acid (DNA) synthesis inhibitors and identical susceptibility to some highly specific bacteriophages. Identical buoyant densities of their DNAs in isopycnic CsCl density gradients and identical thermal denaturation temperatures of their DNAs suggest that clones I-110 and L1-110 have the same DNA base composition. Preliminary DNA/DNA hybridization experiments show that strain I-110 DNA and strain L1-110 DNA have a high degree of common polynucleotide sequences.     

Architecture of Alkaline-soluble and Bioactive Polysaccharide from the Kernels of Prunus mume SIEB. et ZUCC. Assessed by Anti P-1 Antibody

P-1 was partially hydrolyzed with 0.01, 0.05, and 0.1 M trifluoroacetic acid (TFA), successively, and the dialyzable (E-1, E-2, and E-3) and non-dialyzable (I-1,I-2, and I-3) fractions were prepared and analyzed chemically and immunochemically. Either I-1 or E-1 reacted with anti P-1 serum as strongly as P-1 and were mitogenic. The cross-reactivity of I-2 and I-3 was less than I-1 with anti P-1 serum. However, they were as mitogenic as I-1. The cross-reactivity of E-2 and E-3 to anti P-1 serum was also very weak, and they were not mitogenic. The E-1 fraction had a similar sugar composition to I-1 and P-1. E-2 was a monosaccharide, all of Ara, and would be from the linkage of furanosyl residues in P-1. The composition of E-3 was free from Ara and the structure of E-3 was similar to that of I-3. E-3 would be considered to be deleted arabinofuranose from E-1. These results suggest that the mitogenic activity measured by the alkaline phosphatase assay is a property of the core part, I-3, but that P-1 contains several epitopes other than the core part by the immunochemical analysis.     

Phosphorylation of phosphoprotein phosphatase inhibitor-2 (I-2) in rat fat cells

Fat cells were incubated with 32Pi for 2 h before the [32P]I-2 was immunoprecipitated, subjected to SDS/PAGE, and detected by autoradiography. [32P]I-2 (Mr = 32,000) was not recovered when excess purified I-2 was added with the antiserum or when nonimmune serum was used. Immunoprecipitated I-2 was heat-stable, inhibited phosphatase activity, and could be synergistically phosphorylated by casein kinase II and FA/GSK-3. Several times more [32P]phosphoserine than [32P]phosphothreonine was found in I-2 from 32P-labeled cells. Insulin increased the 32P-content of I-2 by as much as 40%, suggesting that phosphorylation of I-2 might be involved in the effect of insulin on stimulating protein dephosphorylation.     

Synthesis and biological evaluation of nucleoside analogues than contain silatrane on the basis of the structure of acyclovir (ACV) as novel inhibitors of hepatitis B virus (HBV)

Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combination principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte was observed after treatment, as evaluated by flow cytometry (FCM).     

Inflorescence bud protease(s) against dehydrin-like inflorescence bud proteins of Pistacia vera

This paper reports on a proteolytic activity extracted from Pistacia vera L. (pistachio) inflorescence buds, found in male and female tissues. The proteolytic activity reaches a peak at the beginning of bud opening and flowering and is directed against inflorescence bud dehydrin-like proteins of pistachio, IBP32 and IBP27. The effect of protease inhibitors indicated that the protease(s) belong to a serine-protease-like family and are not cysteine, acid or metallo-proteases. The proteolytic activity was strongly inhibited by Nα-p-tosyl-L-lysine chloromethyl ketone and to a lesser extent by N-tosyl-L-phenylalanine chloromethyl ketone, suggesting mainly trypsin-like specificity or broader serine-protease specificity. It is suggested that the proteolytic activity is important in the mobilization of nitrogen reserves stored in the bud storage proteins during dormancy to support the fast-developing inflorescence in spring after bud dormancy break.     

Natural plant enzyme inhibitors. Characterization of an unusual alpha-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.).

An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.     

The chaperone-like properties of mammalian inhibitor-2 are conserved in a Drosophila homologue

Phosphatase inhibitor-2 (I-2) is a mammalian phosphoprotein that binds to the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1c) and inhibits its activity in vitro. Recombinant PP1c differs from native PP1c in several biochemical criteria, including the requirement for Mn(2+), sensitivity to vanadate, and p-nitrophenyl phosphate (pNPP) phosphatase activity. I-2 can convert recombinant PP1c into a native-like activity in vitro. It has therefore been suggested that I-2 may act as a molecular chaperone for PP1 in vivo. We have identified a Drosophila homologue (I-2Dm) in a two-hybrid screen for PP1c-binding proteins. The sequence of I-2Dm is 35% identical with that of I-2, whereas the catalytic subunits themselves are >85% identical in flies and humans; however, we show that many biochemical properties of I-2 are conserved. Like I-2, I-2Dm can convert recombinant PP1c to a native-like activity. This strongly suggests that this ability is an essential, conserved role of I-2 and I-2Dm.     

Identification and characterization of a novel protein inhibitor of type 1 protein phosphatase

We have isolated human cDNA for a novel type 1 protein phosphatase (PP1) inhibitory protein, named inhibitor-4 (I-4), from a cDNA library of germ cell tumors. I-4, composed of 202 amino acids, is 44% identical to a PP1 inhibitor, inhibitor-2 (I-2). I-4 conserves functionally important structure of I-2 and exhibited similar biochemical properties. I-4 inhibited activity of the catalytic subunit of PP1 (PP1C), specifically with an IC(50) of 0.2 nM, more potently than I-2 with an IC(50) of 2 nM. I-4 weakly inhibited the activity of myosin-associated phosphates (PP1M). However, the level of inhibition of PP1M was increased during preincubation of PP1M with I-4, suggesting that the inhibition is caused by interaction of I-4 with PP1C in such a manner that it competes with the M subunit of PP1M. Gel overlay experiments showed that I-4 binds PP1C directly. Three I-4 peptides containing the N-terminal residues 1-123, 1-131, and 1-142 all showed strong binding ability to PP1C but did not show PP1 inhibitory activity, whereas an I-2 peptide (residues 1-134), lacking the corresponding C-terminal residues, potently inhibited PP1C activity as previously reported. Removal of the 18 N-terminal amino acid residues from I-4 dramatically reduced the PP1 binding activity with a correlated loss of inhibitory activity, whereas removal of the 10 N-terminal residues had only a little effect. The two peptides GST-I-4(19-131) and GST-I-4(132-202) showed ability to bind to PP1C, albeit very weakly. These results strongly suggest a multiple-point interaction between I-4 and PP1C, which is thought to cause the inhibition of I-4 which is stronger than the inhibition of I-2.     

Identification and domain mapping of Dictyostelium discoideum type-1 protein phosphatase inhibitor-2

The protein phosphatase type-1 catalytic subunit (PP1c) does not exist freely in the cell and its activity must be very strictly controlled. Several protein inhibitors of PP1c have been described including the classical mammalian inhibitor-1 (I-1) and inhibitor-2 (I-2). Association of these inhibitors with PP1c appears to involve multiple contacts and in the case of I-2 no less than five I-2 interaction subdomains have been proposed. In this report, we provide both in vitro and in vivo evidence that the Dictyostelium discoideum genome encodes a protein (DdI-2) that is an ortholog of mammalian I-2, being the first PP1c interacting protein characterized in this social amoeba. Despite the low overall sequence similarity of DdI-2 with other I-2 sequences and its long N-terminal extension, the five PP1c interaction motifs proposed for mammalian I-2 are reasonably conserved in the Dictyostelium ortholog. We demonstrate that DdI-2 interacts with and inhibits D. discoideum PP1c (DdPP1c), which we have previously characterized. Moreover, using yeast two-hybrid assays we show that a stable interaction of DdI-2 with DdPP1c requires multiple contacts.     

Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.     

牛角花齿蓟马为害对紫花苜蓿AsA、GSH含量及相关代谢酶活性的影响

为了明确非酶抗氧化物质抗坏血酸(AsA)、还原型谷胱甘肽(GSH)及相关代谢酶抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)在紫花苜蓿(Medicago sativa L.)对牛角花齿蓟马Odontothrips loti Haliday为害的抗性中的作用,测定了不同牛角花齿蓟马虫口密度下抗、感蓟马苜蓿无性系R-1、I-1的AsA、GSH含量及APX、GR活性的变化。结果表明:受牛角花齿蓟马为害后,R-1无性系在低虫口密度(1、3头/枝条)下,AsA、GSH含量和GR活性均上升,在高虫口密度(5、7头/枝条)下,AsA含量和GR活性先升高后下降,GSH含量上升后保持稳定;I-1无性系的AsA、GSH含量先升高后下降,GR活性在为害后期呈上升趋势;R-1、I-1无性系的APX活性均先上升后下降,但R-1无性系APX活性的上升速率及下降速率小于I-1无性系。说明AsA、GSH含量及APX、GR活性的升高可能是紫花苜蓿对牛角花齿蓟马诱导抗性的一种表现,但I-1无性系对蓟马为害的应激反应滞后于R-1无性系。在牛角花齿蓟马为害后期,R-1无性系体内的AsA、GSH含量及APX、GR活性仍处于较高水平,也说明了R-1无性系对牛角花齿蓟马为害的抗性较I-1无性系强。     

Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).